Treating cancer

ABSTRACT

This document relates to methods and materials involved in treating cancer (e.g., melanoma). For example, methods and materials involved in using an anti-chronic inflammation treatment (e.g., chemotherapy) in combination with a cancer treatment agent (e.g., a cancer vaccine) to treat cancer are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/US2009/049511, filed Jul. 2, 2009, which claims the benefit ofpriority from U.S. Provisional Application Ser. No. 61/078,203, filed onJul. 3, 2008. The disclosures of the prior applications are consideredpart of (and are incorporated by reference in) the disclosure of thisapplication.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in treatingcancer (e.g., melanoma). For example, this document relates to methodsand materials involved in using an anti-chronic inflammation treatment(e.g., chemotherapy) in combination with a cancer treatment agent (e.g.,a cancer vaccine) to treat cancer.

2. Background Information

Cancer is a serious illness that affects many people every year.Melanoma is the most serious form of skin cancer. It is a malignanttumor that originates in melanocytes, the cells which produce thepigment melanin that colors skin, hair, and eyes and is heavilyconcentrated in most moles. While it is not the most common type of skincancer, melanoma underlies the majority of skin cancer-related deaths.About 48,000 deaths worldwide are registered annually as being due tomalignant melanoma. Worldwide, there are about 160,000 new cases ofmelanoma each year. Melanoma is more frequent in white men and isparticularly common in white populations living in sunny climates. Otherrisk factors for developing melanoma include a history of sunburn,excessive sun exposure, living in a sunny climate or at high altitude,having many moles or large moles, and a family or personal history ofskin cancer.

SUMMARY

This document provides methods and materials related to treating cancer.For example, this document provides methods and materials for using ananti-chronic inflammation treatment (e.g., chemotherapy) in combinationwith a cancer treatment agent (e.g., a cancer vaccine) to treat cancer.As described herein, cancer can induce a global state of immunedysfunction and/or chronic inflammation in the cancer patient. Thisglobal state of immune dysfunction and/or chronic inflammation canprevent the patient from mounting a successful response against thecancer. For example, a cancer patient with a global state of chronicinflammation can be in a state such that the patient is unable togenerate an anti-cancer immune response when given an anti-cancervaccine. The methods and materials provided herein can be used to reducethe global state of immune dysfunction and/or chronic inflammationpresent within a cancer patient such that the cancer patient can betterrespond to a cancer treatment such as a cancer vaccine. As describedherein, chemotherapy, radiation, anti-IL-4 agents (e.g., anti-IL-4antibodies), anti-IL-13 agents (e.g., soluble IL-13 receptor), steroids,and combinations thereof can be used to reduce the global state ofimmune dysfunction and/or chronic inflammation present within a cancerpatient. Once the global state of immune dysfunction and/or chronicinflammation present within a cancer patient is reduced, the cancerpatient can be treated with an appropriate cancer treatment such as acancer vaccine or other immune stimulating agents (e.g., IL-2 or IL-12).

In general, this document features a method for treating a mammal havingcancer. The method comprises, or consists essentially of, (a)administering to the mammal an anti-chronic inflammation treatment underconditions wherein the level of global chronic inflammation in themammal is reduced, and (b) administering to the mammal a cancertreatment agent under conditions wherein the presence of the cancer isreduced. The mammal can be a human. The cancer can be melanoma. Thecancer can be stage IV melanoma. The anti-chronic inflammation treatmentcan comprise chemotherapy, radiation, an anti-IL-4 agent, an anti-IL-13agent, or a steroid treatment. The cancer treatment agent can be acancer vaccine. The cancer vaccine can be a MART-1, gp100, or survivincancer vaccine. The period of time between the last administration ofthe anti-chronic inflammation treatment and the first administration ofthe cancer treatment agent can be between two weeks and six months.

In another aspect, this document features a method for treating a mammalhaving cancer. The method comprises, or consists essentially of, (a)administering to the mammal an anti-TGFβ antibody under conditionswherein the level of global chronic inflammation in the mammal isreduced, and (b) administering to the mammal a cancer treatment agentunder conditions wherein the presence of the cancer is reduced. Themammal can be a human. The cancer can be melanoma. The cancer can bestage IV melanoma. The cancer treatment agent can be a cancer vaccine.The cancer vaccine can be a MART-1, gp100, or survivin cancer vaccine.The period of time between the last administration of the anti-TGFβantibody and the first administration of the cancer treatment agent canbe between two weeks and six months.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Effects of 1% patient plasma (vs. normal plasma) on in vitromaturation of normal DC. Presented is a representative experimentdemonstrating the % co-stimulatory molecule positive (CD80, 83, 86) DC.

FIG. 2. Allogeneic mixed lymphocyte reaction cultures evaluatingproliferation of T cells mixed at differing ratios with mature orimmature dendritic cells in the presence of normal (control) plasma orplasma from a patient with metastatic melanoma (MTB plasma). T cellproliferation was assessed by 3H-TdR incorporation. Similar results wereobserved in two other experiments.

FIG. 3. Three dimensional representation of the results of PrincipalComponent Analysis (PCA). The PCA was performed on 234 clinical samplesfor concentrations of 27 cytokines. Each sphere represents one clinicalsample. The dark spheres represent stage IV melanoma patients, and thelighter spheres represent atypical nevi, benign nevi, in situ melanoma,stage I melanoma, stage II melanoma, or stage III melanoma. The axes arethe main principal components. Significant grouping is only evident forpatients in the cohort of stage IV (metastatic) melanoma (dark spheres).

FIG. 4: Mean plasma IL-4 levels (pg/mL)±SD across stages of melanoma(melanoma in situ and stages: I, II, III, IV), patients with atypicalnevi (atypical) and benign nevi (normal controls).

FIG. 5: Assessment of T-cell phenotype and function in healthy donorsand stage IV melanoma patients. The number of T-cells exhibiting theFoxP3 (Treg) or PD-1 phenotype in peripheral blood was determined inhealthy donors and stage IV melanoma patients (A). The frequency ofFoxP3 positive cells were measured by 3-color flow cytometry, CD4-PC5,CD25-PE and FoxP3-Alexa flour 488. The mean percent (+/−SD) of FoxP3positive were determined from the CD4 and CD25 double positivepopulation. The mean frequency (+/−SD) of PD-1+ cells was measured fromthe CD8+ population. The frequency (+/−SD) of tetramer positive (CMV orMART-1) CD8+ T cells was compared among normal volunteers and patientswith stage IV melanoma (B).

FIG. 6: VEGF levels in patients with metastatic melanoma. (A) RNAexpression of cytokines in human metastatic melanoma tissue. Twenty-fourfrozen biopsies of metastatic melanoma tumor tissues was used to extractRNA for expression array analysis. Illustrated are the RNA expressionintensity profiles of 45 probes for 24 cytokines. (B) Comparison ofexpression intensities between genes coding for Th1 (IFN-γ and IL-2),Th2 (IL-4, IL-5, IL-10, and IL-13) cytokines and VEGF. There were nostatistically significant differences when comparing Th1 vs. Th2cytokine expression levels (p=0.04). There was a statisticallysignificant difference when comparing VEGF expression with Th1 or Th2cytokines (p<0.001). Levels of significance were determined using theWilcoxin signed-rank test. (C) ELISA (mean concentration +/−SD) forVEGF-A was performed on plasma samples from healthy donors (n=30) andstage IV melanoma patients (n=40).

FIG. 7. Healthy donor PBMCs were cultured in vitro for 48 hours in thepresence of increasing concentrations of recombinant human VEGFA. At theend of the incubation, cells were stimulated with PMA and ionomycin, inthe presence of brefeldin A and stained for intracellular IFNγ, IL-4, orIL-13 and surface immunophenotyped for CD294 or TIM-3. Cells were thenanalyzed for the frequency of CD4 cells (% of CD4) expressing saidphenotypes using flow cytometry.

FIG. 8: Co-culture with recombinant human VEGF shifts T-helper polarityfrom Th1 (IFN-γ) to Th2 (IL-4) predominance. PBMC (A) isolated fromhealthy donors were stimulated with PMA and ionomycin in the presence ofbrefeldin-A, permeabolized, and intracellularly stained for human IFN-γ(FITC) and human IL-4 (PE). PBMC were exposed to increasingconcentrations of VEGF (0-16 pg/mL) without/with IL-12. All cells wereimmunostained with PC5 anti-human CD4. Purified CD4+ T-cells (B) werenegatively isolated using Miltenyi beads, cultured, and stained in thesame fashion as PBMC (A). Similar results were observed in 5 differentexperiments.

FIG. 9. Changes in plasma VEGF levels (plasma VEGFA in pg/mL; top left)at three time points in a single patient with metastatic melanomatreated on protocol N047a correlate with improved Th1/Th2 ratio asdetermined by intracellular staining of CD4+ cells for IFN gamma or IL-4(top right). These also correlate with emergence of increasedfrequencies of tumor specific CTL (bottom right) as determined bytetramer assay.

FIG. 10. Cellular interactions of acute and chronic inflammation. MSC(myeloid suppressor cell); Th1 & Th2 (T helper lymphocytes type 1 & 2);Treg: regulatory T cell; DC1 (dendritic cells, type 1); DC2 (dendriticcells type 2); CTL (cytotoxic T lymphocyte); illustrated is central roleof tumor derived VEGF in polarizing immunity towards Th2 mediated“chronic inflammation”.

FIG. 11: Correlation of surface immunophenotyping for CD294 and TIM-3with intracellular IL-4, IL-13 and IFNγ for the purposes of enumerationof Th2 and Th1 cells respectively.

FIG. 12: Changes in the ratio of human PBMC derived CD4 T cell subsets(Th1 vs. Th2) following in vitro incubation with varying concentrationsof VEGFA or TGFβ. These results indicate that both VEGFA and TGFβ have asimilar effect on Th1/Th2 polarity in human PBMC derived CD4 cells.

FIG. 13: Relative ratios of human PBMC derived CD4 T cells subsets (Th1vs. Th2) cultured in vitro with varying concentrations of VEGFA in theabsence or presence of increasing concentrations of anti-TGFβneutralizing antibody. Untreated (media) as well as Th1 (Th1) and Th2(Th2) favorable in vitro conditions are presented as controls. Theseresults indicate that presence of anti-TGFβ antibodies reverses theTh1/Th2 modulation of VEGFA in vitro, suggesting that the observed VEGFeffect in these cells may be TGFβ mediated.

DETAILED DESCRIPTION

This document provides methods and materials related to treating cancerin mammals. For example, this document provides methods and materialsrelated to the use of an anti-chronic inflammation treatment (e.g.,chemotherapy) in combination with a cancer treatment agent (e.g., acancer vaccine) to treat cancer.

The methods and materials provided herein can be used to treat cancer inany type of mammal including, without limitation, mice, rats, dogs,cats, horses, cows, pigs, monkeys, and humans. Any type of cancer, suchas skin cancer (e.g., melanoma), can be treated. Examples of cancer thatcan be treated as described herein include, without limitation, skincancer, lung cancer, breast cancer, prostate cancer, ovarian cancer, andcolon cancer. In some cases, stage I, stage II, stage III, or stage IVmelanoma can be treated using the methods and materials provided herein.

In general, cancer can be treated by administering an anti-chronicinflammation treatment such that the global state of immune dysfunctionand/or chronic inflammation present within a cancer patient is reduced.For example, chemotherapy, radiation, anti-IL-4 agents (e.g., anti-IL-4antibodies), anti-IL-13 agents (e.g., soluble IL-13 receptor), steroids,and combinations thereof can be used to reduce the global state ofimmune dysfunction and/or chronic inflammation present within a cancerpatient. In some cases, chemotherapy such as paclitaxel, carboplatin,temozolomide, or cyclophosphamide can be administered to a cancerpatient to reduce the global state of immune dysfunction and/or chronicinflammation present within a cancer patient. In some cases, ananti-chronic inflammation treatment can include, without limitation,administering an anti-TGFβ antibody. For example, anti-TGFβ antibodiescan be administered to a cancer patient to reduce the global state ofimmune dysfunction and/or chronic inflammation present within a cancerpatient. Examples of anti-TGFβ antibodies include, without limitation,human monoclonal anti-TGF-β1 antibodies such as CAT-192 (Genzyme Inc.).

Any appropriate method can be used to assess whether or not the globalstate of immune dysfunction and/or chronic inflammation was reducedfollowing an anti-chronic inflammation treatment. For example, cytokineprofiles (e.g., IL-4, IL-13, IL-4, IL-13, IL-5, IL-10, IL-2, andinterferon gamma) present in blood can be assessed before and after ananti-chronic inflammation treatment to determine whether or not theglobal state of immune dysfunction and/or chronic inflammation wasreduced.

Once the global state of immune dysfunction and/or chronic inflammationpresent within a cancer patient is reduced, the cancer patient can betreated with an appropriate cancer treatment (e.g., an immune cancertreatment) such as a cancer vaccine. Examples of appropriate cancertreatment agents include, without limitation, immune stimulatingcytokines (e.g., IL-2, IL-12, interferon alpha, and interferon gamma),inhibitors of immune down-regulation (e.g., anti-CTLA4, anti-41bb,anti-PD-1, and anti-CD25), and cancer vaccines (e.g., MART-1, gp100,survivin, and tyrosinase cancer vaccines). It will be appreciated thatpaclitaxel, carboplatin, bevacizumab, and anti-CTLA-4 can be used totreat (e.g., skin cancer) upon administration either individually or inany combination thereof (e.g., paclitaxel, carboplatin and bevacizumab).

In some cases, the amount of time between administration of ananti-chronic inflammation treatment and administration of a cancertreatment can be between two weeks and twelve months (e.g., between twoweeks and eleven months, between two weeks and ten months, between twoweeks and nine months, between two weeks and eight months, between twoweeks and seven months, between two weeks and six months, between onemonth and twelve months, between one month and six months, or betweentwo months and six months). For example, a chemotherapy agent (e.g.,paclitaxel) can be administered to a cancer patient to reduce the globalstate of immune dysfunction and/or chronic inflammation present withinthe cancer patient. Then, after one month without any type ofanti-chronic inflammation treatment, a cancer treatment (e.g., a cancervaccine) can be administered to the cancer patient.

Any appropriate method can be used to administer a cancer treatmentagent to a mammal For example, a cancer treatment agent can beadministered orally or via injection (e.g., subcutaneous injection,intramuscular injection, intravenous injection, or intrathecalinjection). In some cases, cancer treatment agents can be administeredby different routes. For example, one cancer treatment agent can beadministered orally and a second cancer treatment agent can beadministered via injection.

In some cases, a cancer treatment agent can be administered followingresection of a tumor. Cancer treatment agent can be administered to amammal in any amount, at any frequency, and for any duration effectiveto achieve a desired outcome (e.g., to increase progression-freesurvival or to increase the time to progression). In some cases, cancertreatment agents can be administered to a mammal having skin cancer toreduce the progression rate of melanoma by 5, 10, 25, 50, 75, 100, ormore percent. For example, the progression rate can be reduced such thatno additional cancer progression is detected. Any method can be used todetermine whether or not the progression rate of skin cancer is reduced.For example, the progression rate of skin cancer can be assessed byimaging tissue at different time points and determining the amount ofcancer cells present. The amounts of cancer cells determined withintissue at different times can be compared to determine the progressionrate. After treatment as described herein, the progression rate can bedetermined again over another time interval. In some cases, the stage ofskin cancer after treatment can be determined and compared to the stagebefore treatment to determine whether or not the progression rate wasreduced.

In some cases, a cancer treatment agent can be administered to a mammalhaving cancer under conditions where progression-free survival or timeto progression is increased (e.g., by 5, 10, 25, 50, 75, 100, or morepercent) as compared to the median progression-free survival or time toprogression, respectively, of corresponding mammals having untreatedcancer.

An effective amount of a cancer treatment agent can be any amount thatreduces the progression rate of cancer, increases the progression-freesurvival rate, or increases the median time to progression withoutproducing significant toxicity to the mammal Typically, an effectiveamount of a cancer treatment agent such as bevacizumab can be from about5 mg/kg/week to about 15 mg/kg/week (e.g., about 10 mg/kg/week). If aparticular mammal fails to respond to a particular amount, then theamount of one or more of the compounds can be increased by, for example,two fold. After receiving this higher concentration, the mammal can bemonitored for both responsiveness to the treatment and toxicitysymptoms, and adjustments made accordingly. The effective amount canremain constant or can be adjusted as a sliding scale or variable dosedepending on the mammal's response to treatment. Various factors caninfluence the actual effective amount used for a particular application.For example, the frequency of administration, duration of treatment, useof multiple treatment agents, route of administration, and severity ofthe cancer may require an increase or decrease in the actual effectiveamount administered.

The frequency of administration can be any frequency that reduces theprogression rate of cancer, increases the progression-free survivalrate, or increases the median time to progression without producingsignificant toxicity to the mammal For example, the frequency ofadministration can be from about once a month to about three times amonth, or from about twice a month to about six times a month, or fromabout once every two months to about three times every two months. Thefrequency of administration can remain constant or can be variableduring the duration of treatment. In addition, the frequency ofadministration of multiple cancer treatment agents can be the same orcan differ. For example, one cancer treatment agent can be administeredthree times during a 28 day period, while a second cancer treatmentagent can be administered one time, and third cancer treatment agent canbe administered two times during the same period. A course of treatmentwith a cancer treatment agent can include rest periods. For example, acancer treatment agent can be administered over a two week periodfollowed by a two week rest period, and such a regimen can be repeatedmultiple times. As with the effective amount, various factors caninfluence the actual frequency of administration used for a particularapplication. For example, the effective amount, duration of treatment,use of multiple treatment agents, route of administration, and severityof the cancer may require an increase or decrease in administrationfrequency.

An effective duration for administering a composition provided hereincan be any duration that reduces the progression rate of cancer,increases the progression-free survival rate, or increases the mediantime to progression without producing significant toxicity to the mammalThus, the effective duration can vary from several days to severalweeks, months, or years. In general, the effective duration for thetreatment of cancer can range in duration from several weeks to severalmonths. In some cases, an effective duration can be for as long as anindividual mammal is alive. Multiple factors can influence the actualeffective duration used for a particular treatment. For example, aneffective duration can vary with the frequency of administration,effective amount, use of multiple treatment agents, route ofadministration, and severity of the cancer.

After administering a composition provided herein to a mammal, themammal can be monitored to determine whether or not the cancer wastreated. For example, a mammal can be assessed after treatment todetermine whether or not the progression rate of cancer was reduced(e.g., stopped). As described herein, any appropriate method can be usedto assess progression and survival rates.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Patients, Methods, and Materials Patients

Blood samples collected from patients with early stage melanoma(melanoma in situ and melanoma stage I, II and III) and benign nevi(atypical/dysplastic nevi) were newly diagnosed patients with noprevious treatment. All patients were tumor free at the time ofperipheral blood collection. Samples from patients with metastaticmelanoma (newly diagnosed, previously untreated) as well as healthyvolunteers/controls were collected under a separate melanoma blood andtissue banking protocol. Both protocols were reviewed and approved foruse in these studies. All biospecimens were collected, processed, andstored in uniform fashion following established standard operatingprocedures. All patients signed an informed consent document. Thepresented study describes data obtained from 113 men and 96 womenranging in age from 21 to 85 (Table 1).

TABLE 1 Study patient population distributed by clinical category, age,sex and assayed immune parameters. Assayed immune parameters Age T-cellTotal mean ± SD Cell Plasma Function RNA Clinical category Patients(range) % female Subset Cytokines Tetramer Assay array Benign Nevi 34 51± 12 68 26 34 7 2 0 (21-71) Atypical/Dysplastic 25 52 ± 16 44 22 16 11 10 (25-84) In situ melanoma 36 61 ± 16 36 30 35 16 3 0 (26-84) Stage I 4554 ± 17 44 36 44 16 4 0 (21-82) Stage II 16 55 ± 17 44 11 12 9 0 0(22-81) Stage III 16 53 ± 19 44 14 16 6 1 0 (23-83) Stage IV 37 56 ± 1443 32 30 27 16 24 (28-85)

Collection of Plasma and Peripheral Blood Mononuclear Cells

Peripheral venous blood (50-90 mL) was drawn into heparinized Vacutainertubes that were processed and separated into plasma and peripheral bloodmononuclear cells (PBMC) following gradient centrifugation usingFicoll-Paque (GE Healthcare Uppsala, Sweden). Plasma was collected andimmediately frozen at −80° C. (1 mL aliquots). PBMC were collected,washed in phosphate buffered saline (PBS), counted, diluted to 1×10⁷/mLand viably frozen in 90% cosmic calf serum (Hyclone Inc. Logan, Utah)and 10% DMSO (Sigma St. Louis, Mo.). All assays were batch-analyzed atthe end of the study.

Immunophenotyping

The following anti-human monoclonal antibodies were used in PBMCimmunophenotyping for flow cytometry: anti-CD3-APC, FITC and PE,anti-CD4-FITC, anti-CD8-PE, anti-CD16 PE, anti-CD56 PE, anti-CD62L APC,anti-CD69 FITC, anti-CD14 FITC, anti-CD16 FITC, anti-CD19 FITC,anti-CD11c APC, anti-CD80 PE, anti-CD83 PE, anti-CD86 PE, anti-CD40 APC,anti-HLA-DR PC5, anti-PD-1 (BD Pharmingen San Jose, Calif.). The humanmonoclonal antibodies anti-CD4 PC5 and anti-CD25 PE were purchased fromBiolegend (San Diego, Calif.) and used in conjunction with anti-humanFoxP3 for the enumeration of T_(reg) cells. The following anti-humanmonoclonal antibodies were used for intracellular staining for flowcytometry: anti-IFNβ FITC, anti-IL-13 PE, anti-IL-4 PE (R and D SystemsMinneapolis, Minn.), and anti-FoxP3 Alexaflour 488 (Biolegend San Diego,Calif.).

Previously frozen PBMC (0.5-1.0 ×10⁶ cells/mL) were thawed and aliquotedinto 96 well rounded bottom plates (100 μL/well). The desired antibodyor antibody pool was added at 5 μL/well. The cells and antibodies wereincubated for 30 minutes at 4° C. and washed twice with 1× PBS (CellgroManassas, Va.), 0.1% BSA and 0.05% sodium azide (Sigma St. Louis, Mo.).Four-color flow cytometry was performed on a LSRII flow cytometer(Becton Dickenson San Jose, Calif.), and Cellquest software (BectonDickenson San Jose, Calif.) was utilized for data analysis. Fordendritic cells, a gate was set on cells, which were HLA-DR⁺ and Lin⁻(CD3, CD14, CD16 and CD19). From this population the percentage ofcells, which were CD11c⁺ and positive for costimulatory molecules (CD80,CD83 and CD86) was determined as previously elsewhere (Fricke et al.,Clin. Cancer Res., 13:4840-8 (2007)). A panel of tumor associatedantigen tetramers, MART-1₂₆₋₃₅, gp100₂₆₄₋₂₇₂, gp100₂₀₉₋₂₁₇, andtyrosinase₃₆₉₋₃₇₇ (Beckman Coulter San Jose, Calif.) were used toenumerate the frequency of tumor antigen specific CD8 positive T-cells.Recall antigens, EBV₂₈₀₋₂₈₈ and CMV₄₉₅₋₅₀₃ (Beckman Coulter San Jose,Calif.) were used as positive controls. For tetramer frequencies, a gatewas set on lymphocytes, which were CD8⁺ and negative for CD4, CD14 andCD 19. Three-color flow cytometry was performed on a LSRII flowcytometer (Becton Dickenson San Jose, Calif.) and Cellquest software(Becton Dickenson San Jose, Calif.) was utilized for data analysis.

Functional enumeration of tumor antigen specific CTL was performed usingan artificial antigen presenting cell method (aAPC) as describedelsewhere (Markovic et al., Clin. Exp. Immunol., 145:438-47 (2006)).Briefly, frozen PBMC were thawed, labeled with the desired tumor antigenpeptide/class I tetramers (Beckman Coulter Fullerton, Calif.) andstimulated for 6 hours with streptavidin coated microbeads (InvitrogenOslo, Norway) loaded with HLA-A2 class I containing tumor antigenpeptides of choice (MART-1, gp100 or tyrosinase) and anti-human CD28 inthe presence of brefeldin A (Sigma, St. Louis, Mo.). After stimulation,the cells were fixed with 2% paraformaldehyde (Sigma, St. Louis, Mo.)and then permeabilized with 0.1% saponin (Sigma, St. Louis, Mo.) in PBS.Cells were then immunophenotyped with anti-human CD4-PC5 or CD8-APC andintracellular staining was done with anti-human IFNγ FITC or IL-4 PE.Four-color flow cytometry was performed with a FACSCaliber and Cellquestsoftware (Becton Dickenson San Jose, Calif.) was utilized for dataanalysis.

Plasma Cytokine, Chemokines and Growth Factor Concentrations

Protein levels for 27 cytokines, chemokines, and growth factors,including IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-12p70, IL-13, IL-15, IL-17, Eotaxin, FGF basic, G-CSF, GM-CSF,IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF, RANTES, TNF-α, and VEGF, weremeasured using the Bio-plex cytokine assay (Bio-rad, Hercules, Calif.)as per manufacturer's instructions. Patient plasma was diluted 1:4 indilution buffer and 50 μL was added to washed, fluorescently dyedmicrospheres (beads) to which biomolecules of interest are bound. Thebeads and diluted patient plasma were incubated for 30 minutes at roomtemperature with agitation. After the incubation the beads were washedin Bio-plex wash buffer and placed in 25 μL of detection antibody andincubated for 30 minutes as described above. After washing, the beadswere placed in streptavidin-PE, incubated, and washed a final time. Thebound beads were resuspended in 125 μL Bio-plex assay buffer and readwith the Luminex plate reader (Bio-rad, Hercules, Calif.). Proteinconcentrations were determined using a standard curve generated usingthe high PMT concentrations with sensitivity from 10-1000 pg/mL.

VEGF Mediated T_(h1)/T_(h2) Polarity

To determine the effect of VEGF on T_(h1) and T_(h2) polarity, PBMC fromhealthy donors were stimulated for 3 days with CD3/CD28 expander beads(Invitrogen Oslo, Norway) with and without increasing doses ofrecombinant VEGF (1-16 pg/mL). Cells were also cultured with 10 μg/mLrecombinant human IL-12 (R and D Systems, Minneapolis, Minn.) or 8 μg/mLof a monoclonal anti-human IL-12 (R and D Systems Minneapolis, Minn.clone #24910). After the culture, the cells were harvested andrestimulated with 50 ng/mL PMA (Sigma, St. Louis, Mo.) and 1 μg/mLionomycin (Sigma, St. Louis, Mo.) in the presence of 10 μg/mL brefeldinA for 4 hours. The cells were then stained with anti-human CD4,anti-human IFN-γ and anti-human IL-4 flow cytometry.

Tumor Tissue RNA Extraction and Microarray

Frozen tissue sections of melanoma biopsies, were examined, regions ofpure tumor with little/no evidence of necrosis or stromal infiltrationwere outlined, scraped off the slides, and used for RNA extraction.Total RNA was isolated from the excised tumor tissue using the QiagenRNA extraction kit (Qiagen Valencia, Calif.). The quality of the RNA wasevaluated by obtaining electropherograms on Agilent 2100 Bioanalyzer andRNA integrity number (RIN) using 2100 Expert software (AgilentTechnologies, Inc. Palo Alto, Calif.). cDNA was prepared from a total of10 μg of RNA. Samples were quantified using standard spectrophotometryusing a Tecan spectrophotometer (Tecan US, Research Triangle Park, N.C.)and considered acceptable if the A260/280 reading was >1.7. The purifiedcDNA was used as a template for in vitro transcription reaction for thesynthesis of biotinylated cRNA using RNA transcript labeling reagent(Affymetrix, Santa Clara, Calif.). Labeled cRNA was then fragmented andhybridized onto the U133 Plus 2.0 array. Appropriate amounts offragmented cRNA and control oligonucleotide B2 were added along withcontrol cRNA (BioB, BioC, and BioD), herring sperm DNA, and bovine serumalbumin to the hybridization buffer. The hybridization mixture washeated at 99° C. for 5 minutes followed by incubation at 45° C. for 5minutes before injecting the sample into the microarray. Then, thehybridization was carried out at 45° C. for 16 hours with mixing on arotisserie at 60 rpm. After hybridization, the solutions were removed,and the arrays were washed and then stained withstreptavidin-phycoerythrin (Molecular Probes, Eugene, Oreg.). Afterwashes, arrays were scanned using the GeneChip Scanner 3000 (Affymetrix,Santa Clara, Calif.). The quality of the fragmented biotin labeled cRNAin each experiment was evaluated before hybridizing onto the U133Aexpression array by both obtaining electropherograms on Agilent 2100Bioanalyzer and hybridizing a fraction of the sample onto test-3 arrayas a measure of quality control. GeneSpring GX 7.3 (AgilentTechnologies, Inc. Santa Clara, Calif.) data analysis software was usedto analyze the results of the microarray experiment. Gene expressionvalues were normalized by the GCRMA algorithm (Bolstad et al.,Bioinformatics, 19:185-93 (2003)).

Statistical Analysis

The majority of samples analyzed in this report were randomly assignedto batches for each laboratory assay due to the fact that all sampleswere not collected/processed at the same time. The randomization wasstratified to assure an even distribution across the stages of diseasefor each batch. The distributions of the results of each run wereexamined, and those that did not appear to be normally distributed weretransformed using either logarithmic or square root transformations. Inorder to look at differences in various parameters between stages ofdisease, analysis was performed utilizing analysis of covariance(ANCOVA), adjusting for age, gender, and batch effects. Results of thisanalysis were summarized by least square means and 95% confidenceintervals for each stage of disease. The p-values presented are thosefrom the overall ANCOVA, which compares the means levels of eachparameter across all stages of disease. P-values <0.05 were consideredto be statistically significant. Due to the magnitude of the cytokinedata from the multiplex assay the data was processed using Partek 6.3software (Partek Inc. St. Louis Mo.) and analyzed using a principalcomponent analysis (PCA) approach. PCA was utilized in an effort tovector space transform a multidimensional data set representing 27variables for each individual patient and group patients based onsimilar cytokine concentrations revealing the internal relationships ofcytokines within patient groups (e.g., per stage of melanoma) in anunbiased way.

Example 2 Systemic Immune Dysfunction

Preliminary results support the notion that systemic immune dysfunctioncan lead to the observed induction of tolerance following peptidevaccination in clinical trials. In this example, normal donor myeloid DCwere exposed to in vitro culture conditions (GM-CSF, IL-4, and CD40L)that lead to their differentiation and maturation (expression ofco-stimulatory molecules) (FIG. 1). The addition of patient plasma tothese experimental conditions resulted in a significant reduction in thenumber of DC expressing key co-stimulatory molecules (maturation),suggesting the presence of a soluble inhibitor(s) of DC maturation.Similar observations, with varying degrees of “suppression” were made inanother nine experiments (nine other samples of patient plasma). Evenwhen normal DCs (mature or immature) were combined with other normaldonor lymphocytes in a mixed lymphocyte culture, the presence of patientvs. normal plasma lead to significantly diminished lymphocyteproliferation (FIG. 2). Thus, factors in the plasma of patients withmetastatic melanoma are interrupting normal immune cell function.

Example 3 Evidence for Th2 Driven Systemic Chronic Inflammation inPatients with Metastatic (Stage IV) Melanoma

The identity of the unknown factor(s) could be a known cytokine. Thus, ascreening study was performed to quantify the plasma concentrations of27 different cytokines (BioRad human 27-plex cytokine panel assaying forplasma concentrations of IL-1β, IL-1rα, IL-2, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, basic FGF, eotaxin,G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF, RANTES, TNF-α,and VEGF) in plasma of over 200 patients with all stages of melanoma(stage I thought IV), melanoma in situ, atypical nevi (possiblepre-malignant lesions) as well as normal controls (patients with benignnevi). Due to the volume of data (27 assays for over 200 subjects),principal component analysis (PCA) was used to identify patterns(groupings) of cytokine data based on clinical subject classifications.The results suggested that the most significant differences in plasmacytokine levels among the different patient diagnostic categories wasdetected in the category of patients with stage IV melanoma/metastaticmelanoma (circled brown spheres in FIG. 3). Closer analysis of the dataindicated that the most significant difference among the cytokines wasnoted for IL-4 (FIG. 4, left), the key regulatory cytokine of the Th2immune response. Statistical comparisons (Student's t-test) of the meancytokine concentrations between patients with metastatic melanoma (stageIV) versus all others depicted a pattern of predominance of other Th2cytokines in addition to IL-4 (IL-10, IL-13, IL-5, eotaxin, IL-9) inpatients with stage IV melanoma (FIG. 4, right). For all of thesecytokines, the pattern of plasma concentrations across stages ofmelanoma was similar to that of IL-4 (no significant changes among anyof the patient cohorts except stage IV melanoma). These results arecomplementary to reports suggesting an increased frequency of Th2 cellsin the blood of patients with advanced cancer (as well as chronicinfections) relative to normal controls (Inagaki et al., Int. J. Cancer,118(12):3054-61 (2006); Matsuda et al., Dis. Colon Rectum, 49(4):507-16(2006); Agarwal et al., Cancer Immunol. Immunother., 55(6):734-43(2006); and Kumar et al., Oncol. Rep., 15(6):1513-6 (2006)).

Preliminary analysis of three random samples from patients with stage IVmelanoma also demonstrated an increased frequency of Th2 cells. Thesedata support the hypothesis of the presence of a state of Th2 mediatedchronic inflammation in patients with metastatic melanoma and offer anexplanation to the observed state of systemic immune dysfunction (e.g.,inability to generate effective immunity following vaccination withcancer vaccines) emulating other clinical conditions characterized byTh2 driven systemic chronic inflammation.

PBMC isolated from patients with benign nevi, atypical (includingdysplastic) nevi, as well as patients with in situ, stage I, II, III orIV melanoma were analyzed by flow cytometry to determine the frequenciesof T, NK, and dendritic (DC) cell subsets. There were not significantdifferences in frequencies of T-cell among stages of melanoma asdetermined by numbers of CD3, CD4 or CD8 positive T-cells (Table 2).Similarly, no significant differences were found in activated T-cells(CD3/CD69), total NK cells (CD16/56⁺, CD3⁻), or most DC subsetparameters. As patients with stage IV melanoma appeared to differsignificantly from all others with regard to plasma cytokine profiles,the cell subset analysis of patients with stage IV melanoma werecompared relative to all others. The analysis revealed no significantdifferences among most parameters (Table 3) with the followingexceptions: (a) the frequency of naïve T-cells (CD3/CD62L⁺) as well asactivated DC (CD11c/CD83⁺) were significantly less in patients withstage IV melanoma; and (b) the frequency of tetramer positive CTL forgp100 and tyrosinase (but not MART-1 or CMV and EBV) were increased inpatients with stage IV melanoma. Due to lack of available biospecimens,T_(h1) and T_(h2) enumeration could not be performed. These datasuggested that there appeared to be some level of “immune activation” inpatients with metastatic melanoma that was different from all othercohorts, and this was consistent with a state of T_(h2) mediated“chronic inflammation.”

TABLE 2 Square root averages of cell subsets with the 95% confidenceinterval (parenthesis). The p-value represents the comparison across allstages of disease. Benign Atypical In-Situ Stage I Stage II Stage IIIStage IV Variable (N = 22) (N = 21) (N = 27) (N = 27) (N = 12) (N = 13)(N = 32) p-value Sqrt CD3 4.78 5.46 5.74 5.29 5.50 5.63 4.94 0.20 (4.20,5.37) (4.88, 6.04) (5.23, 6.25) (4.78, 5.80) (4.79, 6.20) (4.94, 6.32)(3.92, 5.96) Sqrt CD3/4 3.97 4.54 4.57 4.40 4.51 4.64 4.00 0.59 (3.41,4.53) (3.98, 5.09) (4.08, 5.06) (3.91, 4.89) (3.84, 5.19) (3.98, 5.31)(3.02, 4.97) Sqrt CD3/8 2.43 2.82 3.17 2.64 2.60 2.93 2.56 0.20 (1.96,2.89) (2.36, 3.28) (2.77, 3.57) (2.23, 3.04) (2.04, 3.16) (2.39, 3.48)(1.76, 3.37) Sqrt 2.92 2.77 2.91 2.28 2.67 3.15 1.13 0.17 CD3/62L (2.23,3.60) (2.09, 3.45) (2.31, 3.50) (1.69, 2.88) (1.84, 3.49) (2.35, 3.96)(0.00, 2.32) Sqrt CD3/69 0.40 0.54 0.56 0.50 0.50 0.48 0.49 0.95 (0.18,0.61) (0.32, 0.75) (0.37, 0.75) (0.31, 0.69) (0.24, 0.76) (0.23, 0.74)(0.12, 0.87) Sqrt 3.03 3.54 3.44 3.30 3.18 3.43 3.48 0.55 CD3/16 + 56(2.62, 3.44) (3.13, 3.95) (3.08, 3.80) (2.94, 3.66) (2.68, 3.68) (2.95,3.92) (2.77, 4.20) Sqrt 11c+/14− 1.88 2.15 1.89 1.89 2.00 1.98 1.64 0.66(1.59, 2.17) (1.86, 2.44) (1.64, 2.14) (1.64, 2.15) (1.65, 2.35) (1.64,2.32) (1.13, 2.14) Sqrt 11c+/14+ 2.74 3.07 2.60 3.11 2.86 3.50 2.80 0.33(2.18, 3.3) (2.52, 3.63) (2.11, 3.09) (2.62, 3.60) (2.18, 3.54) (2.84,4.16) (1.82, 3.77) Sqrt 11c+/DR 0.80 0.91 0.74 0.87 0.74 0.86 0.78 0.48(0.65, 0.95) (0.77, 1.06) (0.61, 0.87) (0.74, 1.00) (0.56, 0.92) (0.69,1.03) (0.53, 1.03) Sqrt 11c+/DR+ 1.06 1.21 1.06 1.05 1.11 1.01 1.33 0.45(0.88, 1.23) (1.04, 1.39) (0.90, 1.21) (0.90, 1.20) (0.90, 1.32) (0.81,1.22) (1.03, 1.64) Sqrt 11c/80 0.22 0.26 0.24 0.24 0.21 0.25 0.18 0.46(0.17, 0.26) (0.21, 0.30) (0.20, 0.28) (0.20, 0.28) (0.15, 0.26) (0.20,0.31) (0.10, 0.25) Sqrt 11c/83 0.21 0.27 0.24 0.24 0.18 0.19 0.1 0.18(0.15, 0.28) (0.21, 0.33) (0.19, 0.30) (0.18, 0.29) (0.11, 0.26) (0.12,0.26) (0.00, 0.21) Sqrt 11c/86 0.70 0.82 0.71 0.76 0.70 0.75 0.74 0.84(0.56, 0.83) (0.68, 0.95) (0.59, 0.82) (0.64, 0.87) (0.53, 0.86) (0.59,0.91) (0.50, 0.97) Sqrt DR/40 0.54 0.74 0.55 0.62 0.60 0.61 0.82 0.36(0.37, 0.70) (0.58, 0.91) (0.40, 0.69) (0.47, 0.76) (0.40, 0.80) (0.42,0.80) (0.54, 1.11)

TABLE 3 p-value Cytokine (stage IV vs all other) IL-4 1.73 × 10⁻¹²RANTES (CCL5) 6.17 × 10⁻⁰⁶ IL-10 5.29 × 10⁻⁰⁵ Eotaxin (CCL11) 8.31 ×10⁻⁰⁵ IP-10 (CXCL10) 0.0007 IL-13 0.002 IL-12p70 0.005 IL-7, IL-9 0.009VEGF, MIB-1b 0.02 (CCL4) GM-CSF 0.03 IL-5 0.05 IL-15, TNFa,MIP-1a, >0.05 FGF, IL-2, G-CSF, IL- 8, IL-6, IFN-g, MCP-1, IL-17, PDGF,IL-1ra, IL-1b

The emerging data seemed to suggest that patients with stage IVmelanoma, unlike all other patients with earlier stages of melanoma (orhealthy controls), existed in a state of systemic T_(h2) dominance withsome evidence of cellular immune activation in peripheral blood(increased frequencies of tumor specific CTL and decreased frequenciesof naïve T cells). This immune homeostasis profile resembled a state ofT_(h2) dominant “chronic inflammation,” similar to chronic viralinfection (Sester et al., Am. J. Transplant, 5(6):1483-89 (2005)). Areflection of the chronic inflammatory state of chronic viral infectionas well as metastatic melanoma is an increase in peripheral blood PD-1⁺(exhausted) T-cells (Wong et al., Int. Immunol., 19:1223-34 (2007)). Thesame was found to be true in the patient cohort of stage IV melanomapatients compared to healthy controls (FIG. 5 a). This was furthersupported by functional assessment of antigen specific CTL, revealing asignificant reduction in the frequency of functional recall antigen(CMV₄₉₅₋₅₀₃) specific CTL in patients with stage IV melanoma versushealthy volunteers (FIG. 5 b). Less than 5% of tumor antigen specific,PBMC derived, tetramer positive CTL (MART-1) were capable ofintracellular IFN-γ synthesis suggesting immune tolerance.

Example 4 Role of Malignant Melanoma Cells in Tumor-Associated Th₂/IL-4Mediated Systemic Chronic Inflammation

The plasma cytokine profiling data comparing patients across all stagesof melanoma suggested that the greatest differences in the measuredparameters occurred in the setting of metastatic melanoma (stage 4disease). Therefore, it was hypothesized, that the presence of visiblemetastatic disease was in some way responsible for the detected Th₂cytokine dominance in these patients and was likely the result ofmolecules produced by the tumor and/or its interaction with surroundingimmune cells. To that end, the mRNA expression profile of 24 biopsyspecimens of human metastatic melanoma was analyzed looking forup-regulation of expression of known regulatory molecules of immunity(cytokines and chemokines). The mRNA was extracted from frozen sectionsin areas that by H&E staining appeared to contain pure tumor tissue(devoid of necrotic tissue, stroma or lymphocytic infiltrates). The RNAwas analyzed using an Affymetrix U133 plus 2.0 array. In thisexperiment, concurrent blood samples were not available from thepatients for whom tumor tissues existed. The expression of 23 cytokines(45 probes): IL1a and b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IFN-γ, CCL5, CCL11, CSF-2,MCP-1, TNF-α, VEGF was analyzed. The objective of the experiment was todetermine whether or not the malignancy itself was the source of T_(h2)cytokines that were detected in plasma. The data revealed that many ofthe probed cytokines, chemokines, and growth factors are up-regulated intumor tissue (FIG. 6 a). However, there were no differences inexpression of T_(h1) vs. T_(h2) cytokines (IFN-γ vs. IL-4, IL-5, IL-10,and IL-13, FIG. 6 b) in tumor tissues suggesting that the observedT_(h2) cytokine predominance in plasma was not derived from the tumor.However, of the tested cytokines, the most highly/frequentlyup-regulated transcript in the tumor samples was VEGF (FIG. 6 b). PlasmaVEGF levels were significantly higher in metastatic melanoma patientsrelative to healthy donors, (FIG. 6 c), consistent with publishedreports (Tas et al., Melanoma Res., 16:405-11 (2006)). Considering thedescribed immune modulatory (down-regulatory) properties of VEGF(Gabrilovich et al., Nat. Med., 2:1096-103 (1996)), it was postulatedthat tumor derived VEGF could be responsible for the T_(h2) polarizationin patients with stage IV melanoma (away from the normal state of T_(h1)dominance).

VEGF has been associated with DC polarization towards DC₂ leading to Th₂immune responses (e.g., asthma). Likewise, Th₂ cytokines (e.g., IL-4,IL-5, and IL-13) have been associated with increased production of VEGFby a range of different cell types including smooth muscle cells.Preliminary data demonstrated that the addition of recombinant humanVEGFA to a 2-day culture of normal blood-donor derived PBMC appeared toshift Th polarity away from Th₁ and towards Th₂ in a dose dependentfashion (FIG. 7). The addition of VEGF to the cell culture favored areduction of Th cells capable of IFNγ synthesis and TIM-3 expression(Th₁) and increased the frequency of Th cells capable of IL-4 synthesisand CD294 surface expression (Th₂). The effect on intracellular IL13production was less pronounced. Thus, it appeared that VEGFA had adirect impact on human PBMC Th₁/Th₂ polarization in vitro.

In addition, healthy donor PBMC were stimulated with CD3⁺/CD28⁺ expandermicro-beads for 3 days with increasing concentrations (1 pg/mL-16 pg/mL)of recombinant VEGF and assessed intracellular cytokine production ofIL-4 (T_(h2) cytokine) and IFN-γ (T_(h1) cytokine) in CD4⁺ T-cells atthe end of in vitro culture (FIG. 8 a). The data demonstrated thatincreasing concentrations of VEGF resulted in a dose-dependent reversalof the relative ratio of T_(h1) to T_(h2) cells in favor of T_(h2).Increased concentrations of VEGF were associated with a decrease in thenumber of T_(h1) cells (CD4⁺/IFNγ⁺) with an associated reciprocalincrease in T_(h2) cells (CD4⁺/IL-4⁺). The polarizing effects of VEGFwere lost if the assay was performed with purified CD4 cells only (FIG.8 b) suggesting that the observed T_(h) polarization effect of VEGF isindirect, likely mediated by other PBMC. The addition of 10 μg/mL ofIL-12 to the culture containing 16 pg/mL of VEGF prevented the shift inT-helper polarity from T_(h1) to T_(h2); addition of anti-human IL-12antibody to the stimulated PBMC mimicked the effect of VEGF (FIG. 8 a).These data suggest a possible role for monocyte/macrophages in the PBMCpreparation as the mediators of the VEGF induced T_(h) polarization.

To gain further insight into the potential role of tumor produced VEGFon systemic immune homeostasis in vivo in humans with metastaticmelanoma, frozen peripheral blood specimens were randomly selected froma recently completed clinical trial (N047a) where patients withmetastatic melanoma were treated with chemotherapy(paclitaxel+carboplatin) and a specific anti-VEGFA antibody(bevacizumab). They were analyzed for changes in plasma VEGFA levels andTh_(1/)Th₂ polarity as well as frequency of tumor specific CTL (tetramerassay). If VEGF was responsible for Th₂ polarization, its suppressionusing chemotherapy/anti-VEGF therapy would revert the Th₁/Th₂ balanceback to normal (normal ratio is 1:1) and perhaps result in emergence ofnaturally processed anti-tumor specific CTL. Also, this particularclinical trial was chosen because of: (1) available, appropriatelystored biospecimens; and (2) this single arm phase II clinical trialyielded favorable clinical outcomes for patients with metastaticmelanoma, suggesting possible clinical relevance of this therapeuticstrategy. As illustrated in this example from a single patient (FIG. 9),coincident with the decrease of plasma VEGFA concentrations (as a resultof therapy, FIG. 9, top left), there was an increase in Th₁:Th₂ ratioaway from Th₂ and towards Th₁ (FIG. 9, top right) at the same time asthe increase in frequency of tumor specific CTL in peripheral blood(reactive against melanoma differentiation antigens: MART-1, gp100 ortyrosinase, FIG. 9, bottom right). Similar observations were made in twoother patient samples. In all three cases, the increases in tumorspecific CTL tetramer frequency coincided with a reduction in plasmaVEGFA levels to normal levels and a shift in Th polarity away from Th₂and towards Th₁. Of note, such an increase in CTL frequency was notobserved when analyzing the same immunological parameters from patientswith metastatic melanoma treated with chemotherapy alone(nab-paclitaxel+carboplatin) without the anti-VEGF antibody (threepatients analyzed from protocol N057e). Of note, the presented data wereconsistent with a published anecdotal observation from a prior clinicaltrials demonstrating increase in tumor specific CTL tetramer frequencycoincident with reduction of plasma VEGFC levels in patients withmetastatic melanoma treated with a thrombospondin-1 analog, ABT-510(Markovic et al., Am. J. Clin. Oncol., 30(3):303-9 (2007)). Therefore,viewed in the context of the current hypothesis, in addition to theoriginally postulated anti-tumor and anti-angiogenic goals ofpaclitaxel/carboplatin/bevacizumab therapy, the effect of chemotherapy(paclitaxel and carboplatin) may also have depleted (lymphodepleted) thepre-existing state of “chronic inflammation”; and the VEGF inhibitor(bevacizumab) may have allowed reconstitution of tumor-specific immunityin a Th₁ (not Th₂) dominant systemic environment. Thus, it is possiblethat the additional, unanticipated, immunomodulatory effect of thistherapy may have added to the observed therapeutic clinical result.Repeated treatments with lymphodepleting chemotherapy may have alsoinadvertently lead to ultimate depletion of the beneficial anti-tumorimmune response as well, allowing tumor progression. Perhaps, thisexplains in part the clinical outcomes of protocol N047a demonstrating adramatic improvement in median progression free survival (from 6 weeksto 6 months) with a not nearly as significant an improvement in overallsurvival (from 8 months to 12 months).

In summary, preliminary data suggests that patients with advanced(metastatic) melanoma exhibit systemic features of Th₂-mediated chronicinflammation that appears at least in part mediated by tumor-secretedVEGF (FIG. 10). This state of chronic inflammation effectively dampensspontaneously developed anti-tumor CTL immune responses andsignificantly reduces the efficacy of de novo immunization efforts withcancer vaccines/immune modulation in this patient population. Evidenceexists that the observed Th₂/VEGF pathway could be self sustaining andexists in both physiologic (pregnancy) as well as other pathologicstates (e.g., asthma) in humans. Disruption of the Th₂ driven systemicchronic inflammation in patients with advanced melanoma (and possiblyother malignancies) and reconstitution of effective immunity (Th₁dominance) could potentially translate into effective therapy withclinically meaningful results. Therefore, an improved understanding ofthis mechanism of tumor mediated immune dysfunction/tumor progression asa function of Th₂-mediated chronic inflammation could yield therapeutictargets for cancer therapy with agents already in clinical developmentfor Th₂ mediated disorders (e.g., anti-IL-4 antibody).

Example 5 Confirm the Mechanism of Tumor-Induced (VEGF Mediated), Th₂Driven Chronic Inflammation Across Stages of Melanoma Focusing on theFunctional State of cellular Anti-Tumor Immunity

The identified cytokine profiles of plasma suggests the existence of aTh₂ dominant systemic immune environment in the blood of patients withmetastatic melanoma. The analysis of the cellular/functional counterpartof the immune response in these patients across all stages of melanomaremains unknown. To confirm the hypothesis of Th₂ dominant systemicimmunity, the existence of reciprocal, Th₂ polarized, changes in thecellular immune response in these patients across stages of melanomathat will correlate with the described changes in plasma cytokine andVEGF concentrations is determined To address this, one can (a) enumerateTh₁, Th₂ and T_(reg) cells across stages of melanoma; (b); analyze thenumbers and functional/differentiation state of circulating DC (DC₁/DC₂)across stages of melanoma (DC₂ driven Th₂ polarization) and (c) analyzethe functional status (active vs. tolerant) of both tumor-specific (e.g.MART-1, gp100, tyrosinase) and recall antigen specific (EBV, CMV) CTL inthe HLA-A2⁺ subset of patients, across stages of melanoma. These datacan be combined with the existing data on plasma cytokine levels andcorrelated looking for patterns of Th₁ vs. Th₂ cytokine/cellularprofiles across patients and in relation to plasma VEGF levels.

Enumeration of Th₁, Th₂ and T_(reg) cells across stages of melanoma. Inorder to assess whether the Th₂ cytokine predominance in plasma ofpatients with metastatic melanoma is truly a reflection of a systemicimmune polarization towards Th₂ driven chronic inflammation, one canascertain the expected corresponding changes in the frequencies ofcirculating Th cell subsets across disease stage using frozen PMBCsamples corresponding to plasma cytokine samples described above. Theavailable frozen PBMC can be thawed and analyzed for the relativenumbers of Th₁, Th₂ and T_(reg). CD4 cells can be isolated from thawedPBMC specimens using paramagnetic beads coated with anti-CD4 (Dynal,Oslo, Norway), and they can be incubated with mouse-anti-human CD3/CD28coated “stimulator” micro-beads (R and D Systems Minneapolis, Minn.) for6 hours in the presence of 1 ug/ml brefeldin A, (Sigma Aldrich, StLouis, Mo.). After stimulation, the cells can be fixed, permeablized,and stained with APC conjugated mouse anti-human CD4 (Becton Dickinson,San Jose, Calif.) and FITC conjugated mouse anti-human IFNγ andanti-IL-13 (R and D Systems Minneapolis, Minn.). The stained samples canbe analyzed by flow cytometry (FACScan and Cellquest software(Becton-Dickinson, San Jose, Calif.). The results can indicate thepercentage of IFNγ positive/IL-13 (or IL-4) negative (Th₁) and IFNγnegative/IL-13 (or IL-4) positive (Th₂) helper T cells.

GLP validation of anti-CD294 and anti-TIM-3 cell-surface immunostainingcan be completed for the distinction of Th₂ vs. Th₁ cells in ex vivo(unstimulated) frozen PBMC, respectively. Preliminary data suggestedthat CD4/CD294 positive Th₂ cells exclusively produce IL-4 and IL-13 andnot IFNγ upon CD3/CD28 stimulation. Conversely, CD4/TIM-3 positive Th₁cells exclusively produced IFNγ and not IL-4 and IL-13 following thesame in vitro stimulation (FIG. 11). Once validated, the assay can bestandardized and applied to the battery of tests described herein.

Enumeration of T_(reg) can be performed using intracellular staining forFoxP3 of CD4/25 positive lymphocytes Immunophenotyping can be conductedusing commercially available monoclonal antibodies (Biolegend; SanDiego, Calif.). Samples can be analyzed by flow-cytometry by FACScan®and data processed using Cellquest® software (Becton-Dickinson, FranklinLakes, N.J.).

Assessment of peripheral blood DC subset (DC₁/DC₂) andactivation/differentiation status. In order to ascertain whether or notthe state of Th₂ driven systemic chronic inflammation is primarily afunction of systemic DC polarization towards DC₂ (from DC₁) leading toHTL polarization from Th₁ to Th₂ in patients with advanced melanoma, onecan quantify the relative numbers and functional states of DC₁ and DC₂in patients with melanoma across stages of disease. These data can beanalyzed in conjunction with corresponding plasma cytokine/VEGF and Thsubset data (above). Therefore, the available, frozen PBMC correspondingto the plasma cytokine samples described above can be thawed andanalyzed for the relative numbers of DC subsets defined by expression ofCD11c⁺/CD123-(DC₁), and CD11c-/CD123⁺ (DC₂). Each subset can be analyzedfor surface expression of co-stimulatory molecules (CD80, 83, 86)Immunophenotyping can be conducted using commercially availablemonoclonal antibodies (BD Pharmingen; San Jose, Calif.). Samples can beanalyzed by flow-cytometry by FACScan® and data processed usingCellquest® software (Becton-Dickinson, Franklin Lakes, N.J.).

Analysis of the functional status (active/tolerant) of tumor specific(MART-1, gp100, tyrosinase) and recall antigen specific (EBV, CMV) CTLin the HLA-A2⁺ subset of patients across stages of melanoma. Availablefrozen PBMC for the same cell repository as described herein can beanalyzed for the frequency and functional capacity (exhausted, tolerantvs. non-tolerant subsets) of tumor specific and recall antigen specificCTL. If the hypothesis of tumor mediated, Th₂-driven chronicinflammation is correct, the predominant phenotype of the tumor (andrecall) antigen specific CTL can be one of tolerance (inability tosynthesize intracellular IFNγ upon congnant stimulation withtumor-specific peptides) and exhaustion (expression of PD-1). The latterhas already been suggested to be true (Rosenberg et al., J. Immunol.,175(9):6169-76 (2005)) Immunophenotyping of PBMC can be performed usingtetramers for melanoma differentiation antigen specific, HLA-A2 congnantpeptides (MART-1₂₇₋₃₅, gp100₂₀₉₋₂₁₇ and tyrosinase₃₆₈₋₃₇₆) as well as A2cognant peptides of EBV and CMV (Beckman Coulter, San Diego, Calif.).For tetramer analysis, thawed PBMC can be stained with FITC conjugatedanti-CD8, PC5 conjugated anti-human CD4, CD14 and CD19, and conjugatedHLA-A2 tetramers containing peptides from CMV, EBV (controls),MART-1₂₇₋₃₅, gp100₂₀₉₋₂₁₇ and tyrosinase₃₆₈₋₃₇₆. Samples can be analyzedby flow-cytometry and data processed using Cellquest® software(Becton-Dickinson, Franklin Lakes, N.J.). Gates can be set onlymphocytes that were CD4, CD14, and CD19 (PC5) negative and CD8 (APC)positive. Ongoing quality assurance (QA) data suggests an inter-assayvariability with a coefficient of variation (CV) below 5%. Standardcontrol samples can be run alongside all experiments. If the standardcontrol samples generate results beyond ±2SD of the mean, all assayresults can be rejected and the experiment repeated.

Functional analysis of tetramer positive CTL can be performed in patientsamples demonstrating tetramer frequencies of at least 0.1% to melanomadifferentiation or recall antigens. One can proceed to ascertain theability of tetramer positive CTL to synthesize interferon-y (IFNγ) uponstimulation with cognate peptide presented in the context of HLA-A2 andanti-CD28 co-stimulation using artificial antigen presenting cell (aAPC)stimulation. The details of this method are described elsewhere(Markovic et al., Clin. Exp. Immunol., 145(3):438-47 (2006)). In brief,previously frozen patient PBMC can be thawed in batches, labeled with PEconjugated tetramers (Beckman Coulter, San Diego, Calif.), andstimulated for 6 hours, in the presence of 1 mg/mL brefeldin A, (SigmaAldrich, St Louis, Mo.) with pararamagnetic beads (Dynal, Oslo, Norway)coated with peptide loaded HLA-A2 (Beckman Coulter San Diego, Calif.)and mouse anti-human CD28 (R and D Systems Minneapolis, Minn.). Afterstimulation, the cells can be fixed, permeablized, and stained with APCconjugated mouse anti-human CD8 (Becton Dickinson, San Jose, Calif.) andFITC conjugated mouse anti-human IFN-gamma (R and D Systems Minneapolis,Minn.). The stained samples can be analyzed by flow cytometry (FACScanand Cellquest software (Becton-Dickinson, San Jose, Calif.). The resultscan indicate the percentage of tetramer positive CTL able vs. unable tosynthesize intracellular IFNγ. Ongoing QA data suggests an inter-assayvariability with a CV of below 9%. Standard control samples can be runalongside all experiments. If the standard control samples generateresults beyond ±2SD of the mean, all assay results can be rejected, andthe experiment repeated.

Laboratory data summary and statistical analysis. All blood samples wereregistered, collected, processed and annotated. Sample break down perdiagnostic category is described in Table 4.

TABLE 4 Summary of available biospecimens (frozen PBMC and plasma)Clinical diagnostic category Benign Atypical In Situ Stage I Stage IIStage III Stage IV nevi nevi melanoma melanoma melanoma melanomamelanoma Available frozen 26 16 35 36 12 16 30 PBMC with alreadyavailable corresponding plasma cytokine data Additional available frozenPBMC and frozen plasma as of May 1, 2008 Therapeutic 0 0 0 0 0 44 198clinical trials Melanoma blood 10 0 22 43 38 209 421 and tissue bank(MC997 g)

Example 6 Assess the Impact of Systemic Therapeutics on the HypothesizedTumor-Driven Th₂ Mediated State of Systemic Chronic Inflammation WithSpecial Emphasis on the Role of VEGF

The effects of melanoma-specific therapeutic interventions on theVEGF/Th₂ state of tumor-induced chronic inflammation can be examinedusing peripheral blood biospecimens from patients with stage IVmalignant melanoma enrolled on ongoing or completed clinical trials forchanges in a range of immune parameters with a primary focus on Th₁/Th₂balance and functional tumor specific CTL immunity. The trials arelisted in the Table 5. The patients enrolled into these trials had ablood specimen collected before initiation of therapy and after onecycle of treatment. This can provide data on the immediate impact oftherapy on our VEGF/Th2/immune parameters of interest.

TABLE 5 Summary of available biospecimens from therapeutic clinicaltrials for stage IV melanoma Study Number of patients enrolled numberTreatment regimen (accrual as of May 1, 2008) N0377 RAD001² 53(completed) N047a Paclitaxel + Carboplatin + 53 (completed) Bevacizumab¹N057e Abraxane + Carboplatin 74 (completed) MC057f Temozolomide 86 (12)Paclitaxel + Carboplatin 86 (0) N0675 RAD001² + Temozolomide 43 (6)N0775 Abraxane + Carboplatin + 43 (0) Bevacizumab¹ Temozolomide +Bevacizumab 43 (0) ¹Humanized anti-VEGFA antibody; ²rapamycin analog,inhibitor of mTOR (down-regulation of VEGF synthesis)

For each individual patient, the biospecimens collected beforeinitiation of therapy and after one cycle of treatment can be used. Thiscan provide data on the immediate impact of therapy on VEGF/Th2/immuneparameters of interest. Additional testing for later time-points can bepursued only if justified by the initial analysis suggesting beneficialchanges in the studied immune parameters. This can allow one to gaininsight into the effects of a broad range of clinical interventions onimmune homeostasis using an available (but limited) resource ofbiospecimens and only pursue further analysis if justified by thegenerated data.

Laboratory analyses of the stored PBMC can include the same assaysdescribed above and can also include: (a) PBMC immunophenotyping forimmune cell subset analysis; and (b) plasma cytokine profiling.

PBMC immunophenotyping for immune cell subset analysis. Pre andpost-treatment frozen PBMC biospecimens can be analyzed for the “global”impact of therapy on immune cell subsets. One can analyze the relativenumbers of T, B, NK cells, monocytes and DC, and their activation statususing commercially available monoclonal antibodies directed at thefollowing antigens: CD3, CD4, CD8, CD11c, CD14, C16, CD19, CD20, CD25,CD45RA/RO, CD56, CD69, CD63L, CD80, CD83, CD86, CD123, DR, foxP3 (BDPharmingen; San Jose, Calif.) Immunophenotyping can be performed usingmanufacturer's instruction in batch samples of the same patientsanalyzed on the same day. The stained samples can be analyzed by flowcytometry (FACScan and Cellquest software (Becton-Dickinson, San Jose,Calif.). PBMC isolated from patients prior to B7-DC XAb and 15 daysafter antibody treatment can be assayed. Changes in the numbers of cellsbearing the lymphocyte markers in pared comparisons for patients priorto B7-DC XAb treatment can be used to ascertain antibody treatmenteffects.

Plasma cytokine profiling. In order to complement the PBMC derivedcellular immunity analyses, one can add plasma cytokine measurements inthe same samples (paired PBMC and plasma testing). To that end, one canprofile the serum cytokine changes as a result of specific therapy forall available specimens before and after treatment. The BioRad human27-plex cytokine panel can be used (Cat # 171-A11127, Bio-Rad, San DiegoCalif.) for the measurements of plasma concentrations of IL-1β, IL-1rα,IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13,IL-15, IL-17, basic FGF, Eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1,MIP-1α, MIP-1β, PDGF, RANTES, TNF-α, and VEGF. The assay can beperformed as per the manufacturer's directions. Briefly, 100 μL ofBio-Plex assay buffer can be added to each well of a MultiScreen MABVN1.2 μm microfiltration plate followed by the addition of 50 μL of themultiplex bead preparation. Following washing of the beads with theaddition of 100 μL of wash buffer, 50 μL the samples or the standardscan be added to each well and incubated with shaking for 30 minutes atroom temperature. The plasma (1:3 dilution) and standards can be dilutedusing the Bio-Plex human serum diluent kit and plated in duplicate.Standard curves can be generated with a mixture of 27 cytokine standardsand eight serial dilutions ranging from 0-32,000 pg/mL. The plate canthen be washed 3 times followed by incubation of each well in 25 μL ofpre-mixed detection antibodies for 30 minutes with shaking. The platecan further be washed and 50 μL of streptavidin solution were added toeach well and incubated for 10 minutes at room temperature with shaking.The beads can be given a final washing and resuspension in 125 μL ofBio-Plex assay buffer. Cytokine levels in the sera can be quantified byanalyzing 100 μL of each well on a Bio-Plex using Bio-Plex Managersoftware version 4.0. Normal values for plasma cytokine concentrationswere generated by analyzing 30 plasma samples from healthy donors (blooddonors at the Mayo Clinic Dept. of Transfusion Medicine). A set of fivenormal plasma samples (standards) can be run along side all batches ofplasma analysis. If the cytokine concentrations of the “standard”samples differ by more than 20%, results can be rejected, and the plasmasamples re-analyzed.

Example 7 Prospectively Follow Changes of Immune Homeostasis (Evolutionof Systemic Chronic Inflammation) in High-Risk Patients After CompleteResection of Advanced Melanoma Until Subsequent Tumor Relapse

To better understand the clinical relevance of these differences inimmune homeostasis and study the kinetics of their evolution in humansas they develop clinically detectable metastatic cancer (melanoma), onecan perform a prospective clinical trial in which patients withsurgically resected metastatic melanoma (and in a state of chronicinflammation) undergo complete resection of their tumors as part oftheir clinical care and are subsequently followed at regulartime-intervals until tumor relapse. It is hypothesize that followingsurgical resection, the state of systemic “chronic inflammation” willresolve. These patients can then be followed at regular intervals (every2 months), and their blood analyzed for emergence of Th₂-mediatedchronic inflammation, until clinical tumor relapse/recurrence ofmetastatic melanoma (approximately 50% of patients will relapse within18 months of surgery). This study will depict the time-sequence andthresholds of systemic changes in immune homeostasis (chronicinflammation) as they evolve towards the development of relapsedmetastatic melanoma. Sufficient blood specimens (100 mL every 2 months)can be collected to allow complete analysis as well as provide someadditional material for further testing (if necessary). The clinicaltrial can be powered based on the inter-patient variability of the mostprominent immune abnormality in patients with metastatic melanoma (e.g.variability of plasma IL-4 concentrations) determined herein. Ifsuccessful, these data can clinically validate the changes in immunehomeostasis as they impact the natural history of metastatic melanomaand describe potential targets for future therapy. Additionally, as allpatients on this study will undergo surgical resection of metastaticmelanoma as well as undergo concurrent comprehensive immunologicaltesting, their surgical tissues can be processed and preserved foranalysis addressing the influence of the tumor on the observedimmunological profile (multiple frozen blocks for futureimmunohistochemical study and mRNA extraction). All tumor tissue canundergo genomic mRNA expression profiling as well as IHC analysis forinfiltrating immune cell subsets (funded under separate, existinginstruments). These data can correlate the relationship of immunity inthe tumor microenvironment with that of systemic immunity in metastaticcancer.

Study design. The clinical trail can be conducted in the context of theclinical trials program of the Melanoma Study Group of the Mayo ClinicCancer Center. All patients with the diagnosis of metastatic melanomathat are planned to undergo complete surgical resection of theirmalignancy can be offered participation in this study. The objective ofthe study can be to profile the changes in immune homeostasis frompre-surgery, post-surgery and all through the time of clinicallydetectable tumor relapse. It is hypothesized that the state of VEGF/Th₂driven chronic inflammation can be established pre-surgery, resolvedsoon after surgery and slowly re-develop in the months prior to clinicaltumor relapse.

For the purposes of this study, patients can be clinically followed inaccordance to clinical practice (every 2 months). Patients can be askedto donate 100 mL of blood at each follow-up time point. The blood can becollected, processed, and stored in accordance to existing proceduresfor immunological testing Immune homeostasis analysis can be conductedin batches to limit inter-assay variability. Specific focus/priority canbe given to parameters reflecting Th₁/Th₂ balance, frequency offunctional/tolerant tumor (or recall) antigen specific CTL as well asplasma cytokine and VEGF levels. At the time of clinical relapse,patients can be re-tested, and the tumor biopsied for histologicconfirmation. Available tumor tissues can be analyzed for expression oftumor associated antigens (immunohistochemistry for MART-1, gp100 andtyrosinase) as well as tumor infiltrating lymphocytes and compared tothe original surgical specimen for each individual patient. In the rareevents where patients can undergo another curative surgical resection atthe time of relapse, they may continue on study following the outlinedfollow-up/testing schedule until such a time when a tumor relapse is nolonger surgically resectable. See, e.g., Table 6.

Eligibility Criteria.

Required Characteristics/Inclusion Criteria:

1. HLA-A2⁺ adult patients (age 18 years) with metastatic malignantmelanoma who are planned to undergo complete resection for metastaticdisease as part of their regular medical care.2. The following laboratory values obtained 14 days prior toregistration: hemoglobin ≧9.0 g/dL; platelet count ≧75,000/μL; andAST≦3×ULN.3. Ability to provide informed consent.4. Willingness to return to clinic for follow-up.5. ECOG performance status 0, 1 or 2.6. Willingness to participate in the mandatory translational researchcomponent of the study.

Contraindications/Exclusion Criteria:

1. Uncontrolled or current infection.2. Known standard therapy for the patient's disease that is potentiallycurative or proven capable of extending life expectancy.3. Any of the following prior therapies with interval since most recenttreatment: (a) chemotherapy ≦4 weeks prior to registration; or (b)biologic therapy weeks prior to registration.4. Any of the following as this regimen may be harmful to a developingfetus or nursing child: (a) pregnant women; (b) nursing women; or (c)women of childbearing potential or their sexual partners who areunwilling to employ adequate contraception (condoms, diaphragm, birthcontrol pills, injections, intrauterine device (IUD), surgicalsterilization, subcutaneous implants, or abstinence, etc).5. Known immune deficiency or ongoing immunosuppressive therapy.

TABLE 6 Test schedule Every 2 months after ≦14 days prior <7 days priorto surgery until surgically At time of Tests and procedures toregistration scheduled surgery unresectable relapse² tumor relapse²History and exam, X X X weight, performance status Vital signs X XDisease evaluation X X X (clinical/imaging) Hematology group X X X WBC,ALC, ANC, Hgb, platelets Chemistry group X X X AST, LDH, Alk Phos,Creat, K, Na, LDH Immunology studies X^(R) X^(R) X^(R) HLA typing X^(R,)Tumor typing for X^(R) X^(R) MART1, gp100 and tyrosinase; profile ofinfiltrating lymphocytes Serum pregnancy test¹ X^(R) ¹Only for women ofchild-bearing age; ²if a patient has a melanoma relapse that issurgically completely resectable, they may continue on study with thesame follow-up/testing schedule until relapse is no longer surgicallyresectable; ^(R)research funded

Example 8 TGFβ Alters Th1/Th2 Ratios

CD4 T cells (Th1 and Th2 CD4 T cells) derived from human PBMC wereincubated in vitro with varying concentrations of VEGFA (rhVEGFA; 10ng/mL, 50 ng/mL, and 200 ng/mL) or TGFβ (rhTGFβ; 10 ng/mL, 50 ng/mL, and200 ng/mL). After six hours of incubation at 37° C., the ratio of Th1vs. Th2 (Th1/Th2) was determined Both VEGFA and TGFβ exhibited a similareffect on Th1/Th2 polarity in human PBMC derived CD4 cells (FIG. 12).

CD4 T cells (Th1 and Th2 CD4 T cells) derived from human PBMC wereincubated in vitro with VEGFA alone (1 ng/mL, 5 ng/mL, 10 ng/mL, 100ng/mL, or 1000 ng/mL) or VEGFA (100 ng/mL) plus an anti-TGFβ antibody (1ng/mL, 10 ng/mL, 100 ng/mL, 1 μg/mL, 5 μg/mL, or 10 μg/mL). Theanti-TGFβ antibody was obtained from Genzyme Corp. (Cambridge, Mass.).Untreated cells (media only) as well as cells exposed to Th1 or Th2favorable in vitro conditions were used as controls. After six hours ofincubation at 37° C., the ratio of Th1 vs. Th2 (Th1/Th2) was determined.The presence of anti-TGFβ antibodies reversed the Th1/Th2 modulation ofVEGFA in vitro, suggesting that the observed VEGF effect in these cellsmay be TGFβ mediated (FIG. 13).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for treating a mammal having cancer, said method comprising:(a) administering to said mammal an anti-chronic inflammation treatmentunder conditions wherein the level of global chronic inflammation insaid mammal is reduced, and (b) administering to said mammal a cancertreatment agent under conditions wherein the presence of said cancer isreduced.
 2. The method of claim 1, wherein said mammal is a human. 3.The method of claim 1, wherein said cancer is melanoma.
 4. The method ofclaim 1, wherein said cancer is stage IV melanoma.
 5. The method ofclaim 1, wherein said anti-chronic inflammation treatment compriseschemotherapy, radiation, an anti-IL-4 agent, an anti-IL-13 agent, or asteroid treatment.
 6. The method of claim 1, wherein said cancertreatment agent is a cancer vaccine.
 7. The method of claim 1, whereinsaid cancer vaccine is a MART-1, gp100, or survivin cancer vaccine. 8.The method of claim 1, wherein the period of time between the lastadministration of said anti-chronic inflammation treatment and the firstadministration of said cancer treatment agent is between two weeks andsix months.
 9. A method for treating a mammal having cancer, said methodcomprising: (a) administering to said mammal an anti-TGFβ antibody underconditions wherein the level of global chronic inflammation in saidmammal is reduced, and (b) administering to said mammal a cancertreatment agent under conditions wherein the presence of said cancer isreduced.
 10. The method of claim 9, wherein said mammal is a human. 11.The method of claim 9, wherein said cancer is melanoma.
 12. The methodof claim 9, wherein said cancer is stage IV melanoma.
 13. The method ofclaim 9, wherein said cancer treatment agent is a cancer vaccine. 14.The method of claim 9, wherein said cancer vaccine is a MART-1, gp100,or survivin cancer vaccine.
 15. The method of claim 9, wherein theperiod of time between the last administration of said anti-TGFβantibody and the first administration of said cancer treatment agent isbetween two weeks and six months.